Project Summary Gene therapy represents a potential mechanism to restore gene expression patterns in failing organs, particularly in heart failure where downregulated expression of multiple proteins contributes to diastolic failure. Methods for in vivo gene editing have undergone significant development, particularly with novel engineered variants of adeno-associated viral (AAV) vectors for delivery of transgenes or as vehicles for CRISPR/Cas9. However, verification of successful gene editing and longitudinal monitoring of spatiotemporal expression patterns still require invasive biopsies. Biopsies suffer from spatial sampling error, inflict pain, and in patients with heart failure increase the risk of sudden death. Serial non-invasive and multi-organ quantification of the delivery and transduction of gene editing cargo and subsequent transgene expression would provide critical data for further development of gene therapies, and potentially for comprehensive patient monitoring. Magnetic resonance imaging (MRI), which has a large install base throughout the United States, is used as part of routine clinical assessment of cardiac structure and function. An emerging MRI approach termed chemical exchange saturation transfer (CEST) utilizes the endogenous exchange of magnetization between macromolecules and water for in vivo molecular imaging. We have previously developed CEST-MRI methods for non-invasive cardiac imaging of tissue fibrosis, metabolic dysfunction, cell tracking, and most recently to quantify the expression of a genetically encoded 50-Lysine reporter peptide. In this proposal, we seek to develop CEST-MRI methods that exploit the surface Lysine residues of the AAV2 viral capsid protein 3 (VP3) for endogenous CEST-MRI of cellular AAV2 transduction and endosomal escape. Next, we seek to combine such assessment with CEST-MRI of spatiotemporal patterns of transgene expression in the heart and liver alongside corresponding changes in cardiac structure/function. If successful, these methods can be easily implemented on existing clinical MRI scanners, and provide an endogenous mechanism for tracking of gene editing cargo and subsequent multi-scale outcomes without the need for biopsy.